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Abstract

Acute lymphoblastic leukemia (ALL) is
the most common cancer in children. Current treatment strategies for
childhood ALL result in long-term remission for approximately 90% of
patients. However, the therapeutic response is worse among those who
relapse. Several risk stratification approaches based on clinical and
biological aspects have been proposed to intensify treatment in
patients with high risk of relapse and reduce toxicity on those with a
greater probability of cure. The detection of residual leukemic
cells (minimal residual disease, MRD) is the most important prognostic
factor to identify high-risk patients, allowing redefinition of
chemotherapy. In the last decades, several standardized research
protocols evaluated MRD using immunophenotyping by flow cytometry
and/or real-time quantitative polymerase chain reaction at different
time points during treatment. Both methods are highly sensitive (10-3 a 10-5), but expensive, complex, and, because of that, require qualified staff and frequently are restricted to reference centers. The
aim of this article was to review technical aspects of
immunophenotyping by flow cytometry and real-time quantitative
polymerase chain reaction to evaluate MRD in ALL.

Introduction

The incidence of acute lymphoblastic leukemia (ALL) is higher in
childhood and adolescence, and current treatment strategies result in
long-term remission in up to 90% of children affected. However,
therapeutic responses are worse in relapsed patients, indicating the
value of identifying cases at high risk of relapse in order to
intensify treatment and increase the survival.[1,2,3]

Diagnosis
of ALL is based on the identification and quantification of
lymphoblasts by the microscopic evaluation of a bone marrow (BM) sample
and immunophenotypic assessment by flow cytometry, used to define the
affected cell lineage and cell maturation stage and accurately classify
the disease. A diagnostic approach also includes cytogenetic and
molecular analyzes of a BM sample to detect chromosomal and genetic
abnormalities that have prognostic and therapeutic implications.[3-6]

There are clinical and biological factors associated with poor prognosis in children with ALL.[3,7-10]
In the last few decades, several studies have evaluated the presence of
residual leukemic cells at different times after the start of the
treatment as a predictive factor for the adverse evolution of the
disease. Initially, those cells were detected by morphological analysis
of a BM sample, with a cutoff of 5% blast cells defining disease
remission.[4,7,11,12]
Currently, analytical methods with better sensitivity are recommended
that allow the detection of leukemic cells in lower proportions than
those achieved by morphological evaluation, termed minimal residual
disease (MRD).[3,11,13,14]
The occurrence of MRD is now considered the main prognostic indicator
of ALL in children, even in patients with features that suggest a low
risk of relapse, as demonstrated by many studies.[1,7,13,15-18] MRD refines the risk stratification based on traditional features and may be used to redirect treatment.[1,13,19]
An early response to chemotherapy, with rapid reduction of neoplastic
cells, especially at the end of the induction phase, is considered an
important indicator of a favorable evolution and low risk of relapse.[1,9,13,16,19-23]

Treatment
protocols for childhood ALL recommend MRD monitoring at multiple time
points to evaluate the effectiveness of the various stages of
chemotherapy in the elimination of leukemic cells. The evaluations
carried out in the first 3 months of treatment are considered the most
informative for relapse risk stratification. The absence of MRD at the
end of induction therapy is considered the main favorable outcome
predictor1,[16,19,22]
and, an even earlier MRD evaluation – within 2 to 3 weeks of the
initiation of remission induction chemotherapy, has shown additional
benefit in risk stratification.[15,24,25]

In
a study by Conter et al. (2010), 3184 patients with B-ALL, participants
of AIEOP - BFM ALL 2000 multicenter study, were stratified by MRD
measured on days 33 and 78 of treatment, using RQ-PCR. Patients defined
as standard risk (42%) showed a 5-year event-free survival (EFS)
estimated at 92.3 %, while intermediate (52%) and high-risk patients
(6%) showed a 5-year EFS of 77.6 % and 50.1 %, respectively.[1]
Basso et al. (2009) published MRD analysis results by flow cytometry on
day 15 (D15) of treatment of 830 patients who underwent the same
therapeutic protocol and have identified three risk groups - standard
(42%), intermediate (47%) and high (11%), which showed increasing
relapse incidences in five years - 7.5%, 17.5% and 47.2%. In
multivariate analysis, they concluded that the assessment of MRD on D15
of treatment was the main predictor of early relapse and might
complement MRD stratification in later time points.[24]
Due to the slow clearance of leukemic cells in T-ALL, Schrappe et al.
(2011), when evaluating MRD by RQ-PCR in 464 children with T-lineage
ALL, concluded that MRD positivity in D78 is the most important relapse
risk predictor in this group of patients.[18]
Researchers of the Children`s Oncology Group (COG), analyzing data from
2143 children with B-ALL, concluded not only that MRD quantified on D29
of treatment is the most important prognostic factor for patient
outcome, considering all risk groups, but also, that MRD measured in
peripheral blood on D8 by flow cytometry provides additional
information.[16] Data from a study involving 99
children under 1 year indicated that the assessment of MRD (RQ-PCR for
the detection of Ig/TCR genes and MLL rearrangements) allows risk
stratification also in this subgroup of patients with clinical and
prognostic features distinct, and can be used to redefine treatment.[23]

The accurate risk stratification using MRD evaluation requires methodologies that achieve high analytical sensitivity (10-4 - 10-5), enabling the detection of small proportions of residual leukemic cells. Less sensitive techniques (10-2 - 10-3)
allow MRD detection at clinically significant levels, associated with
high risk of relapse, but do not detect patients with lower levels of
MRD, which also have a high risk compared to MRD-negative patients.[26,27]

Due
to the proven association between detectable MRD and higher relapse
risk, various protocols indicate the need to intensify treatment for
children with detectable MRD and to reduce the intensity of
chemotherapy in those who have a rapid response to treatment, with the
objective of reducing toxicity.[1,8,14,15,24,28,29]

Additionally,
assessing MRD offers prognostic information in patients with ALL
relapse who have entered a second remission, and allows prediction of
disease evolution in patients after hematopoietic stem cell transplant
(HSCT).[14,28,30]
A review on this subject, published by Campana et al. (2013), after
analyzing the results of several studies evaluating the MRD prognostic
role in the pre- and post-HSCT periods, concluded that the risk of
disease recurrence after transplantation is significantly higher among
patients with detectable MRD prior to the procedure, as well as MRD
detection in the post-HSCT points to an unfavorable outcome, associated
with higher relapse rates.[31] MRD detection in the
pre-HSCT can also help defining strategies to improve patient outcomes,
such as: establishment of the appropriate time for the procedure,
choice of chemotherapy regimen, and/or use of new drug treatments that
show high effectiveness in the clearance of the residual tumor cells in
patients resistant to conventional chemotherapy.[31]
As an example, it should be noted the use of Blinatumomab, which
represents a new class of anti-CD19 antibody-drug, that redirects T
lymphocytes for selective lysis of tumor cells. A study evaluating the
utilization of this drug in patients with chemotherapy-refractory ALL
and an HSCT indication demonstrated its ability to eradicate resistant
tumor cells in pre-transplant, and its association with higher survival
rates and lower incidence of post-transplant recurrence of the disease.[32]

The
prognostic value of MRD monitoring was also demonstrated in the
therapeutic approach of ALL in adults, helping in the recognition of
high-risk patients who have an indication for HSCT. On the other hand,
those classified as true low risk, with undetectable MRD during
chemotherapy induction/consolidation, may be spared from HSCT and its
associated risks.[33,34]

Detection Methods of MRD

The methodologies, currently available for assessing MRD, allow an average detection of one leukemic cell among 104 to 105 normal cells, which represents a 100-fold increase in sensitivity compared to conventional optical microscopy.[7,12,35,36]
Available methods include: 1) immunophenotyping of neoplastic cells by
flow cytometry, which is aimed at finding cells with aberrant
immunophenotypes of leukemic clones; 2) polymerase chain reaction (PCR)
of the clonal rearrangement regions of T-cell receptor (TCR) and/or
immunoglobulin (Ig) genes; and 3) detection of chimeric transcripts
(mRNA) resulting from chromosomal translocations by reverse
transcription PCR (RT-PCR).[3,37-41]

Chimeric
transcripts arising from chromosomal translocations represent specific
markers of leukemic clones, although only a small proportion of
patients present such alterations, limiting the value of this approach.[4,14,35,38]
As an example, BCR-ABL fusion transcripts can be highlighted. These
transcripts are present in approximately 5% of ALL in children and are
considered highly relevant, due to the association of this finding with
more aggressive disease, that may result in early relapse after a
period of remission.[11,38]

In
this review, the two most frequently used MRD detection methods will be
covered in more detail: immunophenotyping by flow cytometry and
analysis of clonal rearrangements of TCR and Ig by quantitative
real-time PCR (RQ-PCR).[19,35,41,42] Table 1

summarizes the main characteristics of the two methodologies, which will be addressed in the text.

The
search for scientific articles was conducted in PubMed and SciELO
databases, using the following keywords: minimal residual disease,
acute lymphoblastic leukemia, flow cytometry, PCR, and gene
rearrangements of Ig/TCR. Original and review articles published
between 2005 and 2015 were initially selected and, later, relevant
references cited in these items were added.

Table 1. Characteristics of the two most frequently used MRD detection methods*

MRD Evaluation by Flow Cytometry

The use of flow cytometry as an MRD analysis methodology emerged in the late 1980s,[43,44] and its use has been increasing since then, because of further technological and methodological advances.

Immunophenotypic
characterization of leukemic cells at diagnosis provides relevant
information for treatment monitoring by enabling the detection of
residual leukemic cells while allowing classification of the disease
according to the affected cell lineage and cell maturation stage.[5]
For this purpose, it is necessary to build informative panels of
monoclonal antibodies that allow an evaluation of aberrant patterns of
antigen expression, including coexpression of antigens normally
expressed by cells in a different maturation stage (asynchronous
antigen expression); cross or aberrant expression of antigens from
other cell lineages (myeloid, B-lymphoid, or T-lymphoid); and/or
changes in the usual intensity of antigen expression, including
overexpression, low expression, or even loss of expression.[4,5,21,45,46] It is noteworthy that the anomalous antigen expression of blast cells reflects a genetic abnormality in the leukemic clone.[4]

MRD evaluation by flow cytometry achieves a sensitivity of 10-3 to 10-4,
which is lower than the sensitivity achieved by RQ-PCR. It has the
advantages of rapid turnaround time of results, which is especially
important at the beginning of treatment; and broad applicability, since
most ALL present identifiable leukemia-associated immunophenotypes
(LAIP) at diagnosis.[4,14,16,20,33,42,47]
The main limitation of the method is associated with the phenotypic
similarities between leukemic lymphoblasts and nonmalignant
B-lymphocyte precursors, at the stages of bone marrow regeneration
during and after chemotherapy, when false-positive results are more
common. It is worth highlighting the possibility of phenotypic changes
in residual leukemic cells throughout treatment, compared to the
antigen expression pattern at diagnosis.[7,13,33,38,48]
The use of new cell markers and antibody panels with combinations of
four or more fluorochromes is associated with improvement in
sensitivity and specificity of the method.[16,36,38,39,42]
Moreover, accurate and sensitive techniques are technically and
economically feasible in places with limited financial resources.[49,50]

MRD
can be detected by flow cytometry in the early stages of remission
induction chemotherapy, about two weeks after the beginning of the
treatment, using a restricted panel of antibodies, since the detection
of immature cells at this stage indicates the presence of residual
leukemic cells.[15,20,42,51]

However,
distinguishing between residual leukemic cells and nonmalignant
B-lymphocyte precursors in samples collected in phases of chemotherapy
associated with bone marrow recovery (the end of the induction phase of
remission and the consolidation phase of treatment) is a challenge with
this method. Prior knowledge of the standard antigen expression of
lymphoid precursors in different stages of maturation and meticulous
immunophenotyping of the leukemic clone at diagnosis is essential.[21,41,52,53]
Thus, MRD evaluation by flow cytometry at the time mentioned above
points requires the use of a large antibody panel that will allow for
an adequate characterization of the leukemic clone.[28]

Although
there is no consensus on the panel that should be used for this
purpose, different authors recommend working with strategic
combinations of monoclonal antibodies conjugated with four or more
distinct fluorochromes, using a relatively fixed backbone in different
tubes. This strategy involves selecting three or more monoclonal
antibodies that are consistently present in all of the combinations
used and that define lymphoid precursor cells. One or more additional
antibodies are added to each tube, in order to detect aberrant antigen
expression of the leukemic clone.[4,13,21,24,25,29,36,47,48,50,54-56]

The
core set of antibodies used to compound the backbone in the different
combinations useful for MRD detection in B-lineage ALL (B-ALL) usually
include CD45, CD34, CD19, and CD10 - antigen initially named CALLA
(common ALL antigen), expressed with high frequency in childhood B-ALL.[5,13,24,29,36,41,50,54,56]
The additional antibodies should be chosen based on the immunophenotype
of each case at diagnosis. Several studies have reported the
applicability of different markers, among which are the following:
CD123,[12,50,57] CD58,[12,46,50,58-60] CD38,[50,61] CD66c,[46,62,63] CD304,[36,64] CD49f,[65] CD81,[66] and CD11b,[67] as well as aberrant T-lymphoid or myeloid cell markers such as CD2, CD7, CD13, CD15, and CD33.[13] Table 2
describes the antigen expression of the above-described markers that
may be observed in leukemic cells and highlights the markers to be
included in the backbone of the different combinations of monoclonal
antibodies in an MRD detection panel in B-ALL, following the mentioned
strategy.

Table 2. Examples of markers used in MRD
detection by flow cytometry in B-ALL, with a description of the antigen
expression expected pattern and/or possible anomalous antigen
expression of blast cells, in relation to the usual antigen expression
of normal B-cell precursors.

Modulated antigen expression in the early stages of
chemotherapy has been reported, including transient changes in the
intensity of expression of CD10 and CD34 markers.[48,68] To account for
this issue, Irving et al. recommend avoiding the use of predefined
gates and considering, at least, two aberrant immunophenotypes per
patient.[47]

To precisely differentiate between nonmalignant
B-lymphoid precursors, called hematogones, and residual leukemic
precursor cells, it is important to consider the immunophenotype of
normal B-cell precursors in their three different stages of maturation:
early, intermediate, and late. Early B-cell precursors express the CD34
and TdT immaturity markers in combination with CD19, CD38, CD10
(bright), CD22 (weak), and CD45 (intermediate). With maturation, these
cells lose their expression of CD34 and show a progressive reduction in
their expression of CD10, whereas they gain CD20, CD22, and CD45
expression. Surface immunoglobulin acquisition coincides with the gain
of CD20 expression. Mature B-lymphocytes show bright expression of CD22
and CD45, reduced or no expression of CD38, and no expression of CD10.
On the other hand, leukemic lymphoblasts are characterized by the
overexpression of CD10 and reduced or no expression of CD45, in
addition to the asynchronous expression of early and late antigens, and
aberrant expression of other markers.[6,12,52,53,69]

The
immunophenotype of malignant T-lymphoblasts for the most differs
significantly from that of normal marrow and blood T-cells, allowing
easier detection of MRD. The identification of immature T-cells in the
peripheral blood (PB) or BM of T-cell ALL (T-ALL) patients indicates
MRD, as only cells confined to the thymus (thymocytes) should show
these characteristics.[34,65] Table 3
presents commonly used markers in the MRD analysis of T-ALL samples,
including CD3cy (cytoplasm), CD3s (surface), CD7, CD34, TdT, and CD99,
with a description of the antigen expression changes that are
observed.[5,7,12,24,28,46,55,70] The panel could also include other
markers of T-lineage cells, such as CD1a, CD2, CD4, CD5, and CD8,
and/or aberrant B-lymphoid and myeloid markers, such as CD19, CD13, and
CD33, depending on the phenotype determined at
diagnosis.[28,49,70]

Table 3. Commonly used markers in the MRD
analysis by flow cytometry in T-ALL, with a description of the aberrant
antigen expression possibly noted.

In order to allow appropriate determination of residual
leukemic clones, the characterization of a cluster of at least 10
events within a given sample is recommended. Thus, to achieve a
sensitivity of 1×10-4, corresponding to the detection limit of 0.01%, analysis of a minimum of 105
leukocytes is necessary.[4,7,21,25,29,51,56] The proportion of blast
cells should be determined among the total viable nucleated cells,
marked with the nuclear dye SYTO, thus eliminating the inclusion of
anucleated events like erythrocytes, platelets, and
debris.[25,48,51,56,68]

The evaluation of MRD should preferably be
performed with BM samples. MRD levels show a good correlation in PB and
BM in T-ALL; however, the correlation is weak in B-ALL, with lower
levels of MRD in PB.[49,71]

The preparation of PB or BM samples
for analysis might include mononuclear cells isolation by
centrifugation gradient using Ficoll-Hypaque solution.[15,36,45,51,62]
However, the processing of whole blood samples or whole BM has been the
procedure of choice in several studies as it presents the following
advantages: it prevents the selection or arbitrary loss of specific
cell populations; it enables the reliable enumeration of cell
populations present in the sample; it minimizes the chances of
modification of antigen expression; and it reduces the processing time
of the samples.[4,20,37] A study by Luria et al.,[51] comparing these
two ways of sample processing, revealed high correlation coefficients
in samples collected on days 15 (0.875) and 33 (0.82) of treatment.
Gaipa et al.[41] obtained a similar result in an analysis of 266
samples collected on days 15, 33, and 78 of treatment, with 91%
concordant results between the mononuclear cell and total nucleated
cell preparations, using a cutoff value of 0.01%. Higher sensitivity
was observed in the analysis of mononuclear cells, which was attributed
to the greater number of cells acquired.[29] Irving et al. found a
concordance rate of 86% between molecular methods and flow cytometry in
MRD detection in BM samples processed in different ways, including
mononuclear cell preparations for molecular methods and red blood cells
lysis in whole BM samples for immunophenotyping by flow cytometry.[47]

A
study conducted by Dworzak et al., which was aimed for interlaboratory
standardization of flow cytometry assays for MRD detection at multiple
time points in treatment, showed a high concordance of results obtained
in an analysis of 202 samples from four participating centers, with
concordant results in 76% (four centers) and 96% (three centers) of the
samples.[56] In order to understand the discordant results, technical
difficulties inherent to the analysis of samples with normal lymphoid
regeneration, low MRD levels (levels close to the detection limit) and
technical flaws (contamination of tubes and compensation failures, for
example) were considered. Additionally, Luria et al., in a comparative
analysis of the results of two different laboratories, highlighted the
importance of the standardization of data analysis procedures,
revealing that half of the discrepancies between results could be
explained by variations in data interpretation.[51]

Analysis of Clonal Rearrangements of the Ig and TCR Genes by RQ-PCR

Antigen receptor genes (Ig and TCR) include various discontinuous
segments (regions V, variable; D, diversity; and J, junction), which
undergo variable rearrangements during early stages of B and T-lymphoid
cell differentiation. Ig and TCR diversity is generated by a random
joining of a V(D)J exon. During this process, the deletion and/or
random insertion of nucleotides at segment´s junctions can also occur
by forming the so-called N regions. Thus, the regions resulting from Ig
and TCR gene rearrangements represent sequences that are unique to each
lymphocyte.[11,21,35,72-74] In leukemia, Ig and TCR rearrangements can
occur in either B or T-cells. Therefore, rearrangements of the
immunoglobulin heavy chain gene (IgH), light chain kappa (IgK), TCR
delta (TCRD), TCR gamma (TCRG), TCR beta (TCRB) and light chain lambda
(IgL) may be detected at different frequencies in ALL of B and T-cell
lineages.[11,17,19,35,37,41,62,68,72-79]

An analysis of clonal
rearrangements of Ig and TCR genes by PCR, at diagnosis, aims at
finding specific sequences of leukemic clones, usually present in ALL
of T and precursor B-cells, which can be used as a target in MRD
evaluation.[11,72] The sensitivity of PCR assays can vary, depending on
the identified gene rearrangement regions, on the use of specific
primers for individual V, D, and J regions or consensus primers for
conserved regions, on the total amount of DNA analyzed, on the
background identified in normal lymphoid cells, and on the
methodological approach used.[11,72,80]

Conventional PCR methods,
developed in the 1990s, require post-PCR detection techniques
(electrophoresis or dot blotting and hybridization) to identify the
final products of the amplification reaction.[38,72] As an example, the
amplified clonal rearrangements using consensus primers can be
identified based on the size and signal intensity after electrophoretic
separation and subsequent heteroduplex analysis, to distinguish PCR
products derived from monoclonal and polyclonal lymphoid cells. In
follow-up samples, the electrophoretic profiles obtained are compared
with those found at diagnosis. This conventional technique shows a
maximum sensitivity of 0.1%. However, from a methodological point of
view, it is considered relatively simple, fast and low-cost. Although
it does not identify residual leukemic cells in proportions lower than
10-3, it allows the identification of
patients with greater residual tumor burdens and those at high risk of
relapse, and can be considered a cost-effective methodology for MRD
monitoring in countries with limited financial resources.[27,79] A
qualitative MRD result (presence or absence) provides limited
information and does not allow for an evaluation of tumor kinetics,
making it impossible to correlate the final amount of PCR product and
the initial amount of target molecules.[11]

The RQ-PCR technique
represents a significant advance, as it allows the accurate
quantification of a PCR product during the early exponential phase of
the amplification reaction, eliminating the variability of the late
exponential phase and the need for post-PCR manipulation.[35,38,72]
RQ-PCR methods require the design of primers specific for each patient
and, therefore, the additional step of sequencing the clonal
rearrangement identified at diagnosis, and detection of the signal in
follow-up samples is considered specific for the malignant clone.[11]
The detection of Ig and TCR gene rearrangements by RQ-PCR is currently
considered the gold standard for MRD assessment in ALL.[46] It has the
advantages of high analytical sensitivity (10-4 to 10-5),
use of standardized methods, and applicability to most patients with
the disease.[17,19,35,37,38,39,42,76,80] Disadvantages include the high
cost; the difficulty in providing fast results, due to the time
required to design clone-specific primers; and the possibility of
false-negative results due to oligoclonality or new gene rearrangements
during disease.[14,17,35,38] It is noteworthy that the use of this
technique may be restricted to specialized laboratories, due the
complexity of the analyses.[33,35,76]

In the context of the
analysis of Ig and TCR gene rearrangements by PCR methods, it is
important to highlight the relevance of the BIOMED-2 Concerted Action –
a European collaborative study, conducted by van Dongen et al., that
has developed and standardized PCR primer sets for the detection of the
gene rearrangements. Since the completion of the study, all primers and
multiplex tubes are available on a commercial basis.[73]

In
addition to Ig and TCR gene rearrangements, genomic breakpoints that
are secondary to specific translocations, such as rearrangements of the
MLL gene or SIL-TAL fusion genes, represent alternative DNA targets,
although they are less frequently seen.[17,23,38,75]

Summary of the Technical Recommendations for RQ-PCR

Although
BM samples are recommended for the analysis of Ig and TCR clonal
rearrangements by RQ-PCR, PB yields comparable results in T-ALL.[38,81]
The tests should be performed on mononuclear cells separated by
centrifugation gradient, using Ficoll-Hypaque solution, which increases
the sensitivity and reproducibility of the method.[38,75]

For
RQ-PCR analyses, standard procedures are described in the
literature,[17,33,35,37,38,68,72,73,75,76,80] and recommendations for
each step are as follows: 1) DNA amplification by conventional PCR
using consensus primers for the search of Ig and/or TCR gene
rearrangements; 2) Detection of the PCR product by polyacrylamide gel
electrophoresis; 3) Heteroduplex analysis (or gene scanning) for the
differentiation of PCR products derived from monoclonal and polyclonal
lymphoid cells,[82] followed by excision and elution of the band from
the polyacrylamide gel if a homoduplex within the expected size range
is confirmed; 4) Sequencing of the junction regions of the
rearrangements; 5) Comparison to known sequences obtained from
available electronic databases for the identification of the V, D, J
segments involved, and the identification of the N region; 6) Design of
clone-specific primers for the junctional regions; 7) Execution of
RQ-PCR using specific primers for each patient and standard curves
generated from serial dilutions (10-1 to 10-5)
of the sample collected at the initial diagnosis in a DNA pool of
mononuclear cells obtained from 5 to 10 healthy donors, tested in
replicates; 8) Detection of the reaction products by nonspecific
systems (dyes, e.g., SYBR Green I) or specific systems (hydrolysis
probes or hybridization probes conjugated to fluorochromes); 9)
Analysis of the fluorescent signal obtained, based on the fluorescence
intensity of the background, often determined during the first three to
15 PCR cycles – parameter used to calculate the cycle threshold (CT) of
each sample (the PCR cycle at which the fluorescence exceeds the cutoff
for the first time); and 10) Correction for the amount and quality of
DNA by the amplification of control genes in parallel with the test
sample.

Highly
sensitive RQ-PCR assays require accurate identification of the
sequences of the junctional regions of Ig and TCR clonal rearrangements
in each case, which allows the design of specific oligonucleotides.[38]
The specificity of the reaction is assessed by parallel amplification
of a DNA pool control sample obtained from healthy donors. The
sensitivity is defined based on dilution assays that can be performed
with the diagnostic sample or reference materials. The limit of
detection is determined by the last dilution able to generate a
positive signal in the absence of a signal of the polyclonal DNA
control sample, and that can detect each junctional region identified
as a target within the reproducibility range or quantitative range of
the test.[38,72,75,80] In an evaluation of the reproducibility, the
variation in the CT values of the replicates should be less than 1.5 if
the average CT value of the replicates is less than 36. It may be
higher if the average CT value of the replicates is greater. In the
case of nonspecific amplification, the difference in CT values between
specific and nonspecific amplifications must be at least one cycle,
although preferably greater than or equal to three, to minimize false
positive results.[72,75,80] The standard curve, obtained from at least
three dilutions, must present an acceptable slope (between −3.1 and
−3.9) and correlation coefficient (>0.98), according to van der
Velden et al.[80] The quantitative range and the analytical sensitivity
of the test must be determined for the RQ-PCR reaction of the
diagnostic sample to establish the parameters for follow-up samples
from the same patient. If follow-up samples present MRD results out of
the quantitative range of the test, the data should be considered
non-reproducible and, therefore, unable to generate accurate
quantitative results.[80] To interpret the results adequately, the
laboratory report should specify the quantitative range and the
analytical sensitivity of the test.[80]

False
positive results from PCR reactions may be due to the presence of
contaminating DNA or non-specific hybridization to amplified DNA from
normal lymphocytes. False negative results may depend on oligoclonality
or clonal evolution during the disease, leading to the loss of targets
identified at diagnosis.[11,72] Therefore, the use of at least two
Ig/TCR targets per patient is recommended for greater accuracy of MRD
tests.[17,35,37,38,72,80]

Due
to the technical complexity, RQ-PCR assays for MRD detection in ALL
should be performed by reference molecular hematology laboratories that
regularly participate in external quality control programs and
preferably carry out the analysis for a significant number of new cases
per year (minimum of 50 cases).[38,80]

Comparative Studies Between Molecular Techniques and Flow Cytometry

MRD
detection by flow cytometry and/or PCR techniques has been widely used
in studies of childhood ALL. Publications report MRD evaluations using
techniques with different analytical sensitivities, follow-up samples
collected at various times, and groups of patients submitted to
different treatment protocols.[1,13,15,16,19,20,22-24,35,42,54,83] On
the other hand, all of these studies confirm the value of detecting
small numbers of residual leukemic cells to assess the risk of disease
relapse and determine the chemotherapy regimen.Comparative
studies have shown that MRD analyses by flow cytometry and RQ-PCR
methodologies estimate similar levels of residual leukemic cells in
most samples obtained from children with ALL, when present in amounts
greater than 0.01%.[14,19,29,37,47,62,84] With the current techniques,
samples with residual leukemic cells detected by RQ-PCR at levels below
0.01% are often negative by flow cytometry.[28,29] Thus, concordance
between the results obtained by the two methods may depend on the
cutoff used and on the evaluation time.[29,37,84] Gaipa et al., in a
simultaneous analysis of 3,565 BM samples by both methods, at day 15,
day 33, and day 78, found a general concordance rate of 80% using a
cutoff value of 0.01%.[29] However, in an evaluation of the times of
sample collection, concordance between the results obtained at day 33
was lower (70%) than at days 15 and 78 (86% and 87%, respectively). The
discordant results were most often negative by flow cytometry and
positive by RQ-PCR, in samples with low MRD levels (< 0.1%). Using
RQ-PCR as a reference, the sensitivity of flow cytometry was 87% at day
15, decreasing to 47% at day 33 and to 27% at day 78, due to the
progressive reduction in MRD levels during follow-up of patients. The
specificity of flow cytometry was high at all three-time points: 74% at
day 15, 88% at day 33, and 99% at day 78. Similarly, Mejstríková et
al., using a strategy of "predefined gates" observed a higher
concordance between the two methodologies at day 15, when compared to
day 33 and to week 12 of treatment.[46] Malec et al. described similar
results for an analysis of 71 follow-up samples, with 89% concordance
between the two methods, using a cutoff value of 0.01%.[37] Kerst et
al. observed an even higher concordance (97.1%) in a comparative
analysis of 105 follow-up samples, with no indication of the collection
times.[62] Using the same cutoff value, Ryan et al. observed a
qualitative concordance between the methods in 93.8% of 151 samples
analyzed at multiple collection time points over a period of three
years. However, the concordance of the results at day 28 (25 samples)
and in the consolidation phase (weeks 7 to 12, 17 samples) was lower
(84% and 88%, respectively).[84] In order to explain the false RQ-PCR
results, the authors considered the inability to distinguish between
viable and apoptotic cells and the possibility of subclone emergence.
In a view to explain the false results by flow cytometry, they examined
the difficulty of a phenotypic distinction between leukemic cells and
normal B-precursors of the regenerating BM, in addition to the possible
modulation of antigen expression during treatment.

Novel Technologies

A
very sensitive sequencing assay, recently developed and described by
Faham et al., allows the detection and monitoring of all leukemic
rearrangements in a given sample, enabling the detection of clonal
evolution in follow-up samples and reducing false negative results.[85]
This methodology is based on next-generation sequencing (NGS) and uses
consensus primers to amplify all Ig and TCR rearrangements present in
the leukemic clone at diagnosis, allowing their monitoring during
treatment. NGS shows an analytical sensitivity of 10-6,
higher than that achieved by RQ-PCR, and do not demand specific primer
design for each patient, requiring less time to execute. On the other
hand, it has disadvantages such as high complexity and cost.

Conclusions

Despite the extensive literature already available, it is necessary
to evaluate data on MRD as a prognostic factor in ALL for each
therapeutic regimen, considering the differences in the intensity of
treatment protocols, favorable times for evaluation and methodological
differences among the assays.[20,33,35,83,86]It is recommended
that MRD cutoffs for therapeutic decision are defined within each
treatment protocol for ALL, since they depend on the detection method,
the treatment administered before MRD follow-up, the prognostic
stratification of patients and the protocol treatment goals.[33,38] The
cutoff value used by most studies to define MRD positivity is 0.01%,
which is the detection limit of routine tests.[14,19,29] The recent
introduction of high-sensitivity techniques might change the cutoff
point for risk stratification in the near future if very low levels of
MRD are proven to be of clinical value. If properly
standardized, immunological and molecular methods are equally effective
in the detection of clinically significant levels of
MRD.[7,14,19,29,37,47,62,83,84] In general, PCR-based methods are
considered more laborious than immunological methods, and they may have
the additional difficulty to rapidly design clone-specific primers for
early MRD analyses.[42,50]The detection of residual leukemic
cells by flow cytometry in stages of treatment associated with bone
marrow regeneration requires more complex technical validation to
achieve results comparable to those obtained with the RQ-PCR. Thus, it
is possible to use flow cytometry as an alternative or a complement to
the molecular method in monitoring patients undergoing treatment for
ALL. As an example, it is worth mentioning the strategy defined
by Coustan-Smith et al., who chose to use flow cytometry to monitor MRD
during remission induction therapy, reserving Ig and TCR rearrangements
amplification assays for inconclusive cases.[28] Other authors also
suggest the complementary use of the two methodologies, whenever
possible and economically feasible, to accurately stratify patients by
MRD and prevent false negative results due to clonal evolution or
phenotypic changes.[1,7,13,14,29,62,84,86]MRD monitoring during
chemotherapy treatment of childhood ALL is recommended by guidelines
adopted by the main reference institutions of onco-hematology, even in
areas with limited technical and financial resources. MRD detection by
flow cytometry is a viable alternative for services located in such
regions. In contrast, analysis of Ig and TCR gene rearrangements by
RQ-PCR is considered an expensive method, which can limit its use.
Alternatively, some authors have proposed the detection of Ig/TCR
rearrangements by conventional PCR using consensus primers and
homo/heteroduplex analysis, despite its lower analytical sensitivity,
considering that this approach allows identification of patients with
greater residual tumor burden, and then at high risk of
relapse.[22,27,49,50,78,79,87,88]On the other hand, technological
development incorporated by laboratories in regions with greater
resources can facilitate and enhance the assessment of MRD. Thus,
methodologies such as next-generation sequencing and multiparameter
flow cytometry (≥ 8 colors) with automation of data analysis tend to
replace progressively currently available methods.[26,86]

The Mediterranean Journal of Hematology and Infectious Diseases [eISSN 2035-3006]is owned by the U.C.S.C. and it is published by Mattioli 1885, Fidenza, Italy.The MJHID is indexed and abstracted in Science Citation Index Expandedand Journal Citation Reports/InCites beginning with V. 7 (1) 2015.